Chimeric Vaccine for Haemophilus Influenzae-Induced Disease

ABSTRACT

The invention described herein relates to a chimeric protein comprising the NTHi twitching pilus major subunit protein (PilA) presenting a portion of the NTHi OMP P5 protein. The invention provides for vaccine compositions comprising the recombinant chimeric protein and methods of eliciting an immune response using the recombinant chimeric proteins of the invention.

This application claims priority to U.S. Provisional Application No.60/697,642 filed Jul. 8, 2005 and U.S. Provisional Application No.60/801,835 filed May 19, 2006, all of which are incorporated byreference herein in its entirety.

FIELD OF INVENTION

The invention described herein relates to a chimeric protein comprisingthe NTHi twitching pilus major subunit protein (PilA) presenting aportion of the NTHi OMP P5 protein. The invention provides for vaccinecompositions comprising the chimeric protein and methods of eliciting animmune response using the chimeric proteins of the invention.

BACKGROUND

The clinical term for middle ear infections is otitis media (OM).According to Klein, Vaccine, 19 (Suppl. 1): S2-S8, 2000, OM is the mostcommon reason for an ill child to obtain healthcare and for a child inthe United States to receive antibiotics or undergo general anesthesia.Statistics indicate that 24.5 million physician office visits were madefor OM in 1990, representing a greater than 200% increase over thosereported in the 1980s. While rarely associated with mortality, themorbidity associated with OM is significant. Hearing loss is a commonproblem associated with this disease, often affecting a child'sbehavior, education and development of language skills (Baldwin, Am. J.Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol.Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162: 685-694,1990). The socioeconomic impact of OM is also great, with direct andindirect costs of diagnosing and managing OM exceeding $5 billionannually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis. J., 16:S9-11, 1997).

OM is thought to result from infectious, environmental and host geneticsfactors. Bacteria such as Haemophilus influenzae, Streptococcuspneumoniae and Moraxella catarrhalis are the most common infectiousorganisms in OM. Acute OM is a disease characterized by rapid onset andshort duration of signs and symptoms of inflammation in the middle ear,while chronic OM refers to a condition that is defined by the relativelyasymptomatic presence of fluid (or effusion) in the middle ear. However,in chronic OM, despite the absence of certain signs of acute infection(i.e., ear pain or fever), these abnormal middle ear fluids can persistfor periods exceeding three months. Treatment of acute OM by antibiotictherapy is common, but multiple antibiotic-resistant bacteria haveemerged in all three Genera of bacteria responsible for OM. Surgicalmanagement of chronic OM involves the insertion of tympanostomy tubesthrough the tympanic membrane of the ear while a child is under generalanesthesia. While this procedure is commonplace (prevalence rates are ˜1million tubes inserted per year in the U.S. Bright et al., Am. J. PublicHealth, 83(7): 1026-8, 1993) and is highly effective in teens ofrelieving painful symptoms by draining the middle ear of accumulatedfluids, it is invasive and carries incumbent risks (Berman et al.,Pediatrics, 93(3):353-63, 1994; Bright et al., supra.; Cimons, ASM News,60: 527-528; Paap, Ann. Pharmacother., 30(11): 1291-7, 1996). There isthus a need for additional approaches to the management and, preferably,the prevention of OM.

OM vaccine development is most advanced for S. pneumoniae, the primarycausative agent of acute OM (AOM), as evidenced by the recent approvaland release of a seven-valent capsular-conjugate vaccine, PREVNAR®(Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). WhilePREVNAR® has been highly efficacious for invasive pneumococcal disease,coverage for OM has been disappointing (6-8%) with reports of anincreased number of OM cases due to serotypes not included in thevaccine (Black et al., Pedriatr. Infect. Dis J, 19: 187-195, 2000;Eskola et al., Pedriatr. Infect. Dis J., 19: S72-78, 2000; Eskola etal., N. Engl. J. Med., 344: 403-409, 2001; Snow et al., Otol. Neurotol.,23: 1-2, 2002).

H. influenzae is a gram-negative bacterium that, as noted above, plays arole in OM. Clinical isolates of H. influenzae are classified either asserotypes “a” through “f” or as non-typeable depending on the presenceor absence, respectively, of type-specific polysaccharide capsules onthe bacteria. A vaccine for H. influenzae type b has been developed.Like PREVNAR®, the type b H. influenzae vaccines target thepolysaccharide capsule of this organism and thus the vaccine iscomprised of capsule polysaccharide that has been conjugated to aprotein carrier. Neither PREVNAR® or the type b H. influenzae vaccinehave any efficacy for NTHI-induced respiratory tract diseases, includingOM. Less progress has been made for a vaccine for non-typeable H.influenzae (NTHi) which causes approximately 20% of acute OM in childrenand predominates in chronic OM with effusion (Coleman et al., Inf andImmunity, 59(5), 1716-1722, 1991; Klein, Pedriatr. Infect. Dis J., 16,S5-8, 1997; Spinola et al., J. Infect. Dis., 154, 100-109, 1986). NTHican also cause pneumonia, sinusitis, septicemia, endocarditis,epiglottitis, septic arthritis, meningitis, postpartum and neonatalinfections, postpartum and neonatal sepsis, acute and chronicsalpingitis, pericardis, cellulitis, osteomyelitis, endocarditis,cholecystitis, intraabdominal infections, urinary tract infection,mastoiditis, aortic graft infection, conjunctitivitis, Brazilianpurpuric fever, occult bacteremia and exacerbation of underlying lungdiseases such as chronic bronchitis, bronchietasis and cystic fibrosis.A prototype NTHi isolate is the low passage isolate 86-028NP which wasrecovered from a child with chronic OM. This strain has been wellcharacterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5,1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as inchinchilla OM models (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzukiet al., Infect. Immun., 62: 1710-8, 1994; DeMaria et al., Infect.Immun., 64: 5187-92, 1996). The NTHi strain 86-026NP was deposited withthe American Type Culture Collection, 10801 University Blvd., Manassas,Va. 20110, on Oct. 16, 2001 and assigned accession no. PTA-4764. Acontig set from the genome of stain 86-028NP can be found at ColumbusChildren's Research Institute Center for Microbial Pathogenesis website.

Adherence and colonization are acknowledged first steps in thepathogenesis of H. influenzae-induced diseases. As such, H. influenzaeexpress multiple adhesins including hemagglutinating pili, fimbriae andnon-fimbrial adhesins (Gilsdorf et al., Pediatr Res 39, 343-348, 1996;Gilsdorf., Infect. Immun., 65, 2997-3002, 1997; and St. Geme III, Cell.Microbiol., 4, 191-200, 2002). Notably, none of the adhesins describedhave previously been associated with a motility function. Moreover, H.influenzae do not express flagella which are also associated withmotility. Twitching motility is a flagella-independent fowl of bacterialtranslocation over moist surfaces and occurs by extension, tethering,and then retraction of polar structures known as type IV pili (Bardy.,Microbiology, 149, 295-304, 2003; Tonjum and Koomey, Gene, 192, 155-163,1997; Wolfgang et al., EMBO J., 19, 6408-6418,; Mattick, Annu. Rev.Microbiol., 56, 289-314, 2002). Type IV pili are typically 5-7 nm indiameter, several micrometers in length and comprised of a singleprotein subunit assembled into a helical conformation with ˜5 subunitsper turn (Bardy et al., Microbiology, 149, 295-304, 2003; Wall andKaiser, Mol. Microbiol., 32, 1-10, 1999). Type IV pilin subunits areusually 145-160 amino acids in length and may be glycosylated orphosphorylated. There are two classes of pilin subunits, type IVa andtype IVb, which are distinguished from one another by the average lengthof the leader peptide and the mature subunit, which N-methylated aminoacid occupies the N-terminal position of the mature protein, and theaverage length of the D-region (for disulfide region). Most of therespiratory pathogens express class IVa pilins, whereas theenteropathogens more typically express class IVb pilins. Type IVa piliare distinguished by the presence of a highly conserved, hydrophobicN-terminal methylated phenylalanine.

Type IV pili serve as a means of rapid translocation over andcolonization of new surfaces. Thus type IV pilus expression is importantto both adherence and biofilm formation by many bacteria (Mattick, Annu.Rev. Microbiol., 56, 289-314 2002; O'Toole and Kolter, Mol. Microbiol.,30, 295-304, 1998; Klausen et al., Mol. Microbiol., 50, 61-68, 2003;Jesaitis et al., J. Immunol., 171, 4329-4339, 2003), as well asvirulence of Neisseria species, Moraxella bovis, Vibrio cholerae,enteropathogenic Escherichia coli and Pseudomonas aeruginosa, amongothers (O'Toole and Kolter, supra; Klausen et al., supra; Klausen etal., Mol. Microbiol., 48, 1511-1524, 2003; Strom and Lory, Annu. Rev.Microbiol., 47, 565-596, 1993). A biofilm is a complex organization ofbacteria that are anchored to a surface via a bacterially extrudedmatrix, comprised of exopolysaccharide or other substances. The matrixenvelopes the bacteria and protects it from the human immune system.Ehrlich et al., JAMA, 287(13), 1710-1715 (2002) describes biofilmformation by H. influenzae. It has been postulated that blocking theinteraction between type IV pili and the human body can avoid or stopthe bacterial infection (Meyer et al., U.S. Pat. No. 6,268,171 issuedJul. 31, 2001).

Type IV pilus expression is a complex and highly regulated bacterialfunction. In P. aeruginosa, the biogenesis and function of type IV piliis controlled by over forty genes (Strom and Lory, supra). To date, onlya subset of the vast number of related type IV pilus genes (Tonjum andKoomey, supra; Darzins and Russell, Gene, 192, 109-115, 1997) have beenfound in several members of the HAP (Haemophilus, Actinobacillus andPasteurella) family (Stevenson et al., Vet. Microbiol., 92, 121-134,2003; Doughty et al., Vet. Microbiol., 72, 79-90, 2000; Dougherty andSmith, Microbiology, 145, 401-409 1999), but neither expression of typeIV pili nor twitching motility has ever been described for any H.influenzae isolate. In fact, H. influenzae is classically described as abacterium that does not express these structures (Friedrich et al. Appl.Environ. Microbiol., 69, 3695-3700, 2003; Fussenegger et al., Gene, 192,125-134, 1997), despite the presence of a cryptic gene cluster withinthe strain Rd genome (Fleischmann et al., Science, 269, 496-512, 1995).Strain Rd is a non-encapsulated derivative of an H. influenzae serotyped organism (Zwahlen et al., Infect. Immun., 42, 708-715, 1983; Bendlerand Goodgal, J. Microbiol., 70, 411-422, 1972; Risberg et al., Eur. J.Biochem., 261, 171-180, 1999). Although strain Rd has some virulenceproperties, serotype d strains are generally considered to becommensals; they do not frequently cause disease (Daines et al., J. Med.Microbiol., 52, 277-282, 2003). It is therefore important to make thedistinction between disease-causing strains of H. influenzae and strainRd.

Fimbriae, which are surface appendages found on non-typable Haemophilusinfluenzae, are produced by 100% of the bacteria recovered from themiddle ears and nasopharyngeal region of children with chronic otitismedia. A vaccine comprised of fimbrin, a filamentous protein derivedfrom the fimbriae of non-typable Haemophilus influenzae was previouslydeveloped and is useful in studying, preventing, or reducing theseverity of otitis media. However, existing methodologies to isolatefimbrin protein from the bacterial outer membrane are tedious andtime-consuming. Similarly, purification of fimbrin expressed by thefimbrin gene in other host vector, is also tedious due to the homologybetween the fimbrin protein and the outer membrane proteins of the hostvector.

The synthetic chimeric vaccine candidate, denoted as LB1 and describedin U.S. Pat. No. 5,843,464, has shown tremendous efficacy in multiplepre-clinical vaccine trials in two rodent hosts. This synthetic peptidecomprises a B-cell epitope of P5-fimbrin collinearly synthesized with aT-cell promiscuous epitope derived from a fusion protein of the measlesvaccine. Whereas LB1 peptide has been shown to be efficacious inpre-clinical trials, there is concern about the ability to test andmarket a vaccine that contains a T-cell promiscuous epitope for intendeduse in very young children. Therefore, there is a need to developvaccine candidate that elicit a specific and controlled immune responseto H. influenzae.

SUMMARY OF THE INVENTION

The present invention relates to chimeric proteins comprising a portionof the Type IV pilus major subunit protein (PilA) of nontypeable H.influenzae (NTHi) and a portion of NTHi OMP P5 protein (also calledP5-fimbrin, fimbrin or OMP P5-homologous adhesin). In particular, theinvention provides for chimeric proteins comprising PilA modified topresent the B-cell epitope of the LB1 peptide. The invention alsoprovides vaccine compositions comprising one or more chimeric proteinsof the invention and methods of eliciting an immune response using thechimeric proteins of the invention.

The LB1 peptide is a 40 amino acid synthetic chimeric P5-fimbrin derivedpeptide (SEQ ID NO: 53) that induces an immunogenic response to NTHi andis advantageous because it does not require tedious purificationtechniques. The LB1 peptide comprises an N-terminal 19 amino acidpeptide that is a B-cell epitope (SEQ ID NO: 4). The B-cell epitope wasderived from the predicted surface-exposed loop 3 of an outer membraneprotein (fimbrin) of NTHi denoted as OMP P5 (also called P5-fimbrin orOMP P5-homologous adhesin). The LB1 peptide further comprises a short5-mer linker peptide and a 16-residue T cell promiscuous eptiope. The Tcell epitope was derived from a fusion protein of the measles virus. TheT cell promiscuous epitope induces a very strong T cell response inindividuals exposed to this epitope.

The present invention contemplates inserting a portion or fragment ofthe LB1 peptide into a safer and selective carrier protein that does notreduce the effectiveness of inducing a B-cell response. Preferably, theportion of the LB1 peptide is inserted into a carrier that itself alsoconfers protection against NTHi-induced diseases. One such carrier thatmay induce protection against NTHi induced diseases is the protein thatcomprises the NTHi Type IV pilus (twitching pilus) protein, also knownas PilA protein (SEQ ID NO: 2). The PilA protein is encoded by the pilAgene (SEQ ID NO: 1).

The present invention provides for chimeric proteins comprising aportion of the LB1 peptide in order to present the peptide to induce animmunogenic response. The invention contemplates presenting a portion ofthe LB1 peptide that is 12 to 35 amino acids, more preferably presentinga portion of the LB1 peptide that is 15 to 30 amino acids, and mostpreferably presenting a portion of the LB1 peptide that is 18 to 19amino acids and is a subunit of the fimbrin protein. A preferred portionof the LB1 peptide is the N-terminal amino acid sequenceRSDYKFYEDANGTRDHKKG (SEQ ID NO: 4).

In another embodiment, the invention provides for chimeric proteinwherein the PilA protein is modified to present a 24 amino acid peptide.The 24 amino acid peptide may comprise the B-cell epitope of the LB1peptide modified as set out in the amino acid sequence of SEQ ID NO: 5(LVRSDYKFYEDANGTRDHKKGRHT) in which a leucine and valine are added tothe N terminus of the B-cell epitope of LB1 and an arginine, histidineand threonine are at the C terminus of the B-cell epitope of LB1. Thesemodifications to the B-cell epitope are contemplated to assist inprotein folding and/or antigen presentation. The invention furthercontemplates any modifications to the B-cell epitope of LB1 that willassist in protein folding and/or antigen presentation.

The amino acid sequence of the surface exposed loop 3 of NTHi OMP P5 canvary between NTHi strains. The invention contemplates chimeric proteinscomprising a portion of the PilA protein modified to present the B cellepitope of any variant amino acid sequence of loop 3 of the NTHi OMP P5.In particular, the invention provides for chimeric proteins wherein thePilA protein is modified to present one of the following variant NTHiOMP P5 amino acids sequences: RSDYKLYNKNSSSNSTLKNLGE (SEQ ID NO: 6),RSDYKLYNKNSSTLKDLGE (SEQ ID NO: 7) and RSDYKFYDNKRID (SEQ NO: 8). Thevariant peptides also may be presented with a leucine and valine addedto the N terminus and an arginine, histidine and threonine added to theC terminus or any other modification to assist in protein folding and/orantigen presentation.

The chimeric proteins of the invention comprise the modified PilA aminoacids wherein the native PilA amino acids have been substituted with aportion of the LB1 peptide. In addition, the chimeric proteins of theinvention comprise a modified PilA amino acid sequence wherein a portionof the LB1 peptide is inserted within and in addition to the native PilAamino acids. The chimeric proteins of the invention have the ability toinduce the formation of antibodies directed against two proteins andtherefore are more effective and more specific vaccine candidates.

In one embodiment, the chimeric proteins comprise the mature amino acidsequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 2)wherein a portion of the LB1 peptide is inserted between the cysteineresidues at positions 62 and 72 of SEQ ID NO: 2 and may substitute thenative amino acids, such as the chimeric protein having the amino acidsequence of SEQ ID NO: 54. This chimeric protein comprises residues40-149 of SEQ ID NO: 2 and has the B-cell epitope of LB1 (SEQ ID NO: 5)inserted between residues 62 and 72 of SEQ ID NO: 2. In anotherembodiment, the portion of the LB1 peptide is inserted between thecysteine residues at positions 131 and 144 of SEQ ID NO: 2 and maysubstitute the native amino acids such as the protein having the aminoacid sequence of SEQ ID NO: 55. This chimeric protein comprises residues40-149 of SEQ ID NO: 2 and has the B-cell epitope of LB1 (SEQ ID NO: 5)inserted between residues 131 and 144 of SEQ ID NO: 2.

In another embodiment, the chimeric proteins comprise the mature aminoacid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 2)wherein the portion of the LB1 peptide is inserted at the C-terminus ofthe PilA protein. For example, the chimeric protein of SEQ ID NO: 56comprises residues 40-149 of SEQ ID NO: 2 and the B-cell epitope of LB1(SEQ ID NO: 5) is inserted following residue 149 of SEQ ID NO: 2.

In another embodiment, the chimeric proteins comprise the mature aminoacid sequence (residues 13-149) of NTHi PilA protein (SEQ ID NO: 2)wherein the portion of the LB1 peptide is inserted at the N-terminus ofthe PilA protein. For example, the chimeric protein of SEQ ID NO: 57comprises residues 40-149 of SEQ ID NO: 2 and the B-cell epitope of LB1(SEQ ID NO: 5) is inserted before residue 40 of SEQ ID NO: 2.

In a further embodiment, the invention provides for chimeric proteinscomprising a portion of the NTHi PilA protein and one or more of the LB1peptides described herein. The chimeric proteins of the inventioninclude those which present the same LB1 peptide more than once within aportion of the NTHi PilA protein and those which present two or moredifferent LB1 peptides within a portion of the NTHi PilA protein.

The invention further provides for chimeric proteins comprising aportion of the NTHi PilA protein and any antigenic protein that willelicit an immune response.

The NTHi Type IV Pilus (PilA) Polynucleotides and Polypeptides of theInvention

The chimeric proteins of the invention may comprise the full length or aportion of the major subunit of the NTHi Type IV Pilus which is encodedby the gene pilA. The PilA protein of the NTHi isolate 86-028NP isencoded by the nucleic acid sequence set out as SEQ ID NO: 2, which isdescribed in U.S. patent application Ser. No. 11/019,005, incorporatedby reference herein in its entirety. Also provided are polynucleotidesencoding PilA polypeptides from NTHi clinical isolates 1728MEE, 1729MEE,3224A, 10548MEE, 1060MEE, 1885MEE, 1714MEE, 1236MEE, 1128MEE and 214NP.The amino acid sequences of these PilA polypeptides are set out in SEQID NOS: 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52 respectively. Thepossibility of alternative codon usage is specifically contemplated inpolynucleotides encoding the polypeptides. In one embodiment, thepolypeptides are respectively encoded by the nucleotide sequences setout in SEQ ID NOS: 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51.

The invention provides for polynucleotides that hybridize understringent conditions to (a) the complement of the nucleotide sequencesset out in SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51; (b)a polynucleotide which is an allelic variant of any polynucleotidesrecited above; (c) a polynucleotide which encodes a species homolog ofany of the proteins recited above; or (d) a polynucleotide that encodesa polypeptide comprising a specific domain or truncation of thepolypeptides of the present invention. PilA polynucleotides from othernon-typeable H. influenzae strains and from H. influenzae strains a, b,c, e and f are specifically contemplated. These polynucleotides can beidentified and isolated by techniques standard in the art such ashybridization and polymerase chain reaction using part or all of thepolynucleotides of SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and51 as probes or primers, respectively.

The polynucleotides of the invention also include nucleotide sequencesthat are substantially equivalent to the polynucleotides recited above.Polynucleotides according to the invention can have, e.g., at least 65%,at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% andeven more typically at least 95%, 96%, 97%, 98% or 99% sequence identityto the NTHi polynucleotides recited above.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequence fragments that hybridize under stringentconditions to the NTHi nucleotide sequences of SEQ ID NOS: 1, 33, 35,37, 39, 41, 43, 45, 47, 49 and 51, or complements thereof, whichfragment is greater than about 5 nucleotides, preferably 7 nucleotides,more preferably greater than 9 nucleotides and most preferably greaterthan 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides ormore that are selective for (i.e., specifically hybridize to any one ofthe PilA polynucleotides of the invention) are contemplated. Thesenucleic acid sequence fragments capable of specifically hybridizing toan NTHi PilA polynucleotide of the invention can be used as probes todetect NTHi PilA polynucleotides of the invention and/or candifferentiate NTHi PilA polynucleotides of the invention from otherbacterial genes, and are preferably based on unique nucleotidesequences.

The term “stringent” is used herein to refer to conditions that arecommonly understood in the art as stringent. Hybridization stringency isprincipally determined by temperature, ionic strength, and theconcentration of denaturing agents such as formamide. Examples ofstringent conditions for hybridization and washing are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodiumchloride, 0.0015M sodium citrate, and 50% formamide at 42° C. SeeSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also be used,however, the rate of hybridization will be affected. In instanceswherein hybridization of deoxyoligonucleotides is concerned, additionalexemplary stringent hybridization conditions include washing in 6×SSC0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-baseoligos).

Other agents may be included in the hybridization and washing buffersfor the purpose of reducing non-specific and/or backgroundhybridization. Examples are 0.1% bovine serum albumin, 0.1%polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodiumdodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt's solution, sonicatedsalmon sperm DNA (or other non-complementary DNA), and dextran sulfate,although other suitable agents can also be used. The concentration andtypes of these additives can be changed without substantially affectingthe stringency of the hybridization conditions. Hybridizationexperiments are usually carried out at pH 6.8-7.4, however, at typicalionic strength conditions, the rate of hybridization is nearlyindependent of pH. See Anderson et al., Nucleic Acid Hybridisation: APractical Approach, Ch. 4, IRL Press Limited (Oxford, England).Hybridization conditions can be adjusted by one skilled in the art inorder to accommodate these variables and allow DNAs of differentsequence relatedness to form hybrids.

As noted above, polynucleotides contemplated by the present inventionare not limited to the specific PilA polynucleotides of SEQ ID NOS: 1,33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, but also include, forexample, allelic and species variations thereof. Allelic and speciesvariations can be routinely determined by comparing the sequenceprovided in SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51,preferably the open reading frames therein, a representative fragmentthereof, or a nucleotide sequence at least 90% identical, preferably 95%identical, to the open reading frames within SEQ ID NOS: 1, 33, 35, 37,39, 41, 43, 45, 47, 49 and 51 with a sequence from another isolate ofthe same species or another species. Preferred computer program methodsto determine identity and similarity between two sequences include, butare not limited to, the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith-Waterman algorithm may also be used to determine identity.

Polynucleotides of the invention may be isolated from natural sources ormay be synthesized by standard chemical techniques, e.g., thephosphotriester method described in Matteucci et al., J Am Chem. Soc.,103: 3185 (1981).

The invention provides for chimeric proteins comprising a portion ofNTHi PilA protein. In one embodiment the polypeptides comprise the NTHi86-028NP amino acid sequences respectively set out in SEQ ID NO: 2.Polypeptides of the invention also include PilA polypeptides set out inSEQ ID NOS: 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52. In additionalembodiments, the PilA polypeptides of the invention are those of othernon-typeable H. influenzae strains and from H. influenzae strains a, b,c, e and f.

Polypeptides of the invention specifically include peptide fragments(i.e., peptides) or fragments of the PilA polypeptide that retain one ormore biological or immunogenic properties of a full length polypeptideof the invention. In one embodiment, PilA peptide fragments provided bythe invention are designated TfpQ2, TfpQ3, TfpQ4 and OLP3 andrespectively comprise amino acids 35 through 68 of SEQ ID NO: 2, aminoacids 69 through 102 of SEQ ID NO: 2, amino acids 103 through 137 of SEQID NO: 2, and amino acids 21 through 35 of SEQ ID NO: 2. Another PilApeptide fragment provided by the invention comprises amino acids 40through 149 of SEQ ID NO: 2.

The invention also provides for chimeric proteins comprising a portionof a PilA polypeptide with one or more conservative amino acidsubstitutions that do not affect the biological and/or immunogenicactivity of the PilA polypeptide. Alternatively, the PilA polypeptidesof the invention are contemplated to have conservative amino acidssubstitutions which may or may not alter biological activity. The term“conservative amino acid substitution” refers to a substitution of anative amino acid residue with a normative residue, including naturallyoccurring and normaturally occurring amino acids, such that there islittle or no effect on the polarity or charge of the amino acid residueat that position. For example, a conservative substitution results fromthe replacement of a non-polar residue in a polypeptide with any othernon-polar residue. Further, any native residue in the polypeptide mayalso be substituted with alanine, according to the methods of “alaninescanning mutagenesis”. Naturally occurring amino acids are characterizedbased on their side chains as follows: basic: arginine, lysine,histidine; acidic: glutamic acid, aspartic acid; uncharged polar:glutamine, asparagine, serine, threonine, tyrosine; and non-polar:phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline,methionine, leucine, norleucine, isoleucine General rules for amino acidsubstitutions are set forth in Table 1 below.

TABLE 1 Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asn Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Phe, Leu Leu Norleucine, Ile, Val, Met, Leu Lys Arg, 1,4Diaminobutyric Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr ArgPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu

The invention also provides for chimeric proteins comprising a portionof a variants of the NTHi PilA polypeptides of the present invention(e.g., a polypeptide exhibiting at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, 86%, 87%,88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at leastabout 95%, 96%, 97%, more typically at least about 98%, or mosttypically at least about 99% amino acid identity to a polypeptide of SEQID NOS: 2, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52) that retainbiological and/or immunogenic activity.

The invention contemplates that PilA polynucleotides of the inventionmay be inserted in a vector for amplification or expression. Forexpression, the polynucleotides are operatively linked to appropriateexpression control sequences such as promoter and polyadenylation signalsequences. Further provided are host cells comprising polynucleotides ofthe invention. Exemplary prokaryotic host cells include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia.Methods of producing polypeptides of the invention by growing the hostcells and isolating polypeptide from the host cells or growth medium arespecifically contemplated. Alternatively, polypeptides of the inventioncan be prepared by chemical synthesis using standard means. Particularlyconvenient are solid phase techniques (see, e.g., Erikson et al., TheProteins (1976) v. 2, Academic Press, New York, p. 255). Automated solidphase synthesizers are commercially available. In addition,modifications in the sequence are easily made by substitution, additionor omission of appropriate residues. For example, a cysteine residue maybe added at the carboxy terminus to provide a sulfhydryl group forconvenient linkage to a carrier protein, or spacer elements, such as anadditional glycine residue, may be incorporated into the sequencebetween the linking amino acid at the C-terminus and the remainder ofthe peptide.

The term “isolated” refers to a substance removed from, and essentiallyfree of, the other components of the environment in which it naturallyexists. For example, a polypeptide is separated from other cellularproteins or a DNA is separated from other DNA flanking it in a genome inwhich it naturally occurs.

Recombinant PilA protein (rPilA) may be generated to serve as a morereadily renewable product. To do this, the published protocol of Keizeret al. (J. Biol. Chem., 276: 24186-14193, 2001), who studied a pilinwhich also had four Cys residues as it will be critical that rPilAsimilarly be properly folded so as to possess functional qualities ofthe native pilin subunit, is utilized. Briefly, a truncated pilin isengineered wherein the first 28 residues are removed from the N-terminusto prevent aggregation, and this truncated pilin will be furtherengineered to be transported to the periplasm by means of theincorporation of an OmpA leader sequence in the construct. Using thisstrategy Keizer et al. generated a recombinant soluble monomeric P.aeruginosa pilin protein that was able to bind to its receptor (asialoGM1) in in vitro assays and decrease morbidity and mortality in micewhen the peptide was delivered 15 minutes prior to heterologouschallenge. This soluble, monomeric, truncated form of NTHi PilA will beuseful in the studies described herein.

The invention also provides for synthetic chimeric proteins. Thechimeric proteins may be synthesize, purified and sequenced usingstandard techniques. For example, the chimeric proteins may be assembledsemi-manually by stepwise Fmoc-tert-butyl solid-phase synthesis andpurified by HPLC. The composition and amino acid sequence of recombinantand synthetic chimeric proteins may be confirmed by amino acid analysisand/or mass spectral analysis.

Antibodies

The invention provides antibodies which bind to antigenic epitopes ofthe chimeric proteins of the invention. The antibodies may be polyclonalantibodies, monoclonal antibodies, antibody fragments which retain theirability to bind their unique epitope (e.g., Fv, Fab and F(ab)2fragments), single chain antibodies and human or humanized antibodies.Antibodies may be generated by techniques standard in the art usingchimeric protein(s) of the invention or host cells expressing chimericprotein(s) of the invention as antigens.

The present invention provides for antibodies specific for the chimericproteins of the present invention and fragments thereof, which exhibitthe ability to kill both H. influenzae bacteria and to protect humansfrom infection. The present invention also provides for antibodiesspecific for the chimeric proteins of the invention which reduce thevirulence, inhibit adherence, inhibit biofilm formation, inhibittwitching motility, inhibit cell division, and/or inhibit penetrationinto the epithelium of H. influenzae bacteria and/or enhancephagocytosis of the H. influenzae bacteria.

In vitro complement mediated bactericidal assay systems (Musher et al.,Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51:31-38, 1972) may be used to measure the bactericidal activity ofanti-chimeric proteins antibodies.

It is also possible to confer short-term protection to a host by passiveimmunotherapy via the administration of pre-formed antibody against achimeric protein of the invention. Thus, antibodies of the invention maybe used in passive immunotherapy. Human immunoglobulin is preferred inhuman medicine because a heterologous immunoglobulin may provoke animmune response to its foreign immunogenic components. Such passiveimmunization could be used on an emergency basis for immediateprotection of unimmunized individuals subject to special risks.

In another embodiment, antibodies of the invention may be used in theproduction of anti-idiotypic antibody, which in turn can be used as anantigen to stimulate an immune response against the chimeric proteinepitopes or H. influenzae epitopes.

Methods for Eliciting an Immune Response and Compositions Therefor

The invention contemplates methods of eliciting in an individual animmune response to H. influenzae in an individual. In certainembodiments, the methods elicit an immune response to the chimericproteins of the invention. These methods elicit one or more immuneresponses, including but not limited to, immune responses which inhibitbacterial replication, immune responses which block H. influenzaeadherence to cells, immune responses which prevent H. influenzaetwitching, immune responses that kill H. influenzae bacteria and immuneresponses which prevent biofilm formation. In one embodiment, themethods comprise a step of administering an immunogenic dose of acomposition comprising one or more chimeric proteins of the invention.In another embodiment, the methods comprise administering an immunogenicdose of a composition comprising a cell expressing one or more chimericproteins of the invention. In yet another embodiment, the methodscomprise administering an immunogenic dose of a composition comprisingone or more polynucleotides encoding one or more chimeric proteins ofthe invention. The polynucleotide may be a naked polynucleotide notassociated with any other nucleic acid or may be in a vector such as aplasmid or viral vector (e.g., adeno-associated virus vector oradenovirus vector). The methods may be used in combination in a singleindividual. The methods may be used prior or subsequent to H. influenzaeinfection of an individual. The methods and compositions of theinvention may be used to treat or prevent any pathological conditioninvolving H. influenzae (typeable and nontypeable strains) such as OM,pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septicarthritis, meningitis, postpartum and neonatal infections, postpartumand neonatal sepsis, acute and chromic salpingitis, epiglottis,pericardis, cellulitis, osteomyelitis, endocarditis, cholecystitis,intraabdominal infections, urinary tract infection, mastoiditis, aorticgraft infection, conjunctitivitis, Brazilian purpuric fever, occultbacteremia, chronic obstructive pulmonary disease and exacerbation ofunderlying lung diseases such as chronic bronchitis, bronchietasis andcystic fibrosis.

In one embodiment of methods of the invention, a composition of theinvention is administered as a priming dose followed by one or morebooster doses. Co-administration of proteins or polypeptides thatbeneficially enhance the immune response such as cytokines (e.g., IL-2,IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) orco-stimulatory molecules is also contemplated.

An “immunogenic dose” of a composition of the invention is one thatgenerates, after administration, a detectable humoral (antibody) and/orcellular (T cell) immune response in comparison to the immune responsedetectable before administration or in comparison to a standard immuneresponse before administration. The invention contemplates that theimmune response resulting from the methods may be protective and/ortherapeutic. In a preferred embodiment, the antibody and/or T cellimmune response protects the individual from H. influenzae infection,particularly infection of the middle ear and/or the nasopharynx or lowerairway. In this use, the precise dose depends on the patient's state ofhealth and weight, the mode of administration, the nature of theformulation, etc., but generally ranges from about 1.0 μg to about 5000μg per 70 kilogram patient, more commonly from about 10 to about 500 μgper 70 kg of body weight.

Humoral immune response may be measured by many well known methods, suchas Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (ETA)and Hemagglutination Inhibition Assay (HAI). In particular, SRIDutilizes a layer of a gel, such as agarose, containing the immunogenbeing tested. A well is cut in the gel and the serum being tested isplaced in the well. Diffusion of the antibody out into the gel leads tothe formation of a precipitation ring whose area is proportional to theconcentration of the antibody in the serum being tested. ETA, also knownas ELISA (Enzyme Linked Immunoassay), is used to determine totalantibodies in the sample. The immunogen is adsorbed to the surface of amicrotiter plate. The test serum is exposed to the plate followed by anenzyme linked immunoglobulin, such as IgG. The enzyme activity adherentto the plate is quantified by any convenient means such asspectrophotometry and is proportional to the concentration of antibodydirected against the immunogen present in the test sample. HAI utilizesthe capability of an immunogen such as viral proteins to agglutinatechicken red blood cells (or the like). The assay detects neutralizingantibodies, i.e., those antibodies able to inhibit hemagglutination.Dilutions of the test serum are incubated with a standard concentrationof immunogen, followed by the addition of the red blood cells. Thepresence of neutralizing antibodies will inhibit the agglutination ofthe red blood cells by the immunogen. Tests to measure cellular immuneresponse include determination of delayed-type hypersensitivity ormeasuring the proliferative response of lymphocytes to target immunogen.

The invention correspondingly provides compositions suitable foreliciting an immune response to chimeric proteins of the invention. Asnoted above, the compositions comprise one or more chimeric proteins,cells expressing one or more chimeric proteins, or one or morepolynucleotides encoding one or more chimeric proteins. The compositionsmay also comprise other ingredients such as carriers and adjuvants.

In compositions of the invention, a chimeric protein may be fused toanother protein when produced by recombinant methods. In one embodiment,the other protein may not, by itself, elicit antibodies, but itstabilizes the first protein and forms a fusion protein retainingimmunogenic activity. In another embodiment, the fusion proteincomprises another protein that is immunogenic, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the fusion protein and facilitateproduction and purification thereof. The other protein may act as anadjuvant in the sense of providing a generalized stimulation of theimmune system. The other protein may be fused to either the amino orcarboxy terminus of the chimeric proteins of the invention.

In other compositions of the invention, chimeric proteins may beotherwise linked to carrier substances. Any method of creating suchlinkages known in the art may be used. Linkages can be formed withhetero-bifunctional agents that generate a disulfide link at onefunctional group end and a peptide link at the other, such as adisulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio)proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982)and bifunctional coupling agents that form a thioether rather than adisulfide linkage such as reactive esters of 6-maleimidocaproic acid,2-bromoacetic acid, 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, andcoupling agent which activate carboxyl groups by combining them withsuccinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt suchas succinimmidyl 4-(N-maleimido-methyl)cyclohexane-1-carobxylate (SMCC).

The chimeric proteins may be formulated as neutral or salt forms.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the peptide) and which are faintedwith inorganic acids such as, e.g., hydrochloric or phosphoric acids, orsuch organic acids as acetic, oxalic, tartaric, mandelic. Salts formedwith the free carboxyl groups may also be derived from inorganic basessuch as, e.g., sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, and procaine.

Compositions of the invention may further comprise adjuvants. Knownadjuvants include, for example, emulsions such as Freund's Adjuvants andother oil emulsions, Bordetella pertussis, MF59, purified saponin fromQuillaja saponaria (QS21), aluminum salts such as hydroxide, phosphateand alum, calcium phosphate, (and other metal salts), gels such asaluminum hydroxide salts, mycobacterial products including muramyldipeptides, solid materials, particles such as liposomes and virosomes.Examples of natural and bacterial products known to be used as adjuvantsinclude monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-likeacylated monosaccharide), OM-174 which is a lipid A derivative from E.coli, holotoxins such as cholera toxin (CT) or one of its derivatives,pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of itsderivatives, and CpG oligonucleotides. Adjuvant activity can be affectedby a number of factors, such as carrier effect, depot formation, alteredlymphocyte recirculation, stimulation of T-lymphocytes, directstimulation of B-lymphocytes and stimulation of macrophages.

Compositions of the invention are typically formulated as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients, which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, e.g., water, saline, dextrose, glycerol, ethanol, or thelike and combinations thereof. In addition, if desired, the vaccine maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, or adjuvants, which enhance theeffectiveness of the vaccine. The vaccines are conventionallyadministered parenterally, by injection, for example, eithersubcutaneously or intramuscularly.

Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25-70%.

Compositions may also be administered through transdermal routesutilizing jet injectors, microneedles, electroporation, sonoporation,microencapsulation, polymers or liposomes, transmucosal routes andintranasal routes using nebulizers, aerosols and nasal sprays.Microencapsulation using natural or synthetic polymers such as starch,alginate and chitosan, D-poly L-lactate (PLA), D-polyDL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters,polyanhydrides and polyphosphazenes polyphosphatazanes are useful forboth transdermal and transmucosal administration. Polymeric complexescomprising synthetic poly-ornithate, poly-lysine and poly-arginine oramphipathic peptides are useful for transdermal delivery systems. Inaddition, due to their amphipathic nature, liposomes are contemplatedfor transdermal, transmucosal and intranasal vaccine delivery systems.Common lipids used for vaccine delivery includeN-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methylsulfate (DOTAP), dioleyloxy-propyl trimethylammonium chloride DOTMA,dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE),dimethyldioctadecyl ammonium bromide (DDAB) and9N(N′,N-dimethylaminoethane)carbamoyl) cholesterol (DC-Chol). Thecombination of helper lipids and liposomes will enhance up-take of theliposomes through the skin. These helper lipids include dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine(DLPE), dimyristoyl phosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoidglycosides or saponins derived from the Chilean soap tree bark (Quillajasaponaria) and chitosan (deacetylated chitan) have been contemplated asuseful adjuvants for intranasal and transmucosal vaccine delivery.

Formulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use.

Methods of Inhibiting H. influenzae

Alternatively, the invention includes methods of inhibiting H.influenzae type IV pili function in an individual. The methods compriseadministering to the individual, for example, one or more antibodies ofthe invention and/or one or more chimeric proteins of the invention; inan amount that inhibits function of the pili. In vitro assays may beused to demonstrate the ability to inhibit pili function. Embodiments ofthese methods include, for example, methods using inhibitors ofadherence mediated via type IV pili, inhibitors that disrupt existingbiofilms mediated by type IV pili, and inhibitors of twitching.

Inhibition is contemplated for any pathological condition involving H.influenzae, for example, OM, pneumonia, sinusitis, septicemia,endocarditis, epiglottitis, septic arthritis, meningitis, postpartum andneonatal infections, postpartum and neonatal sepsis, acute and chromicsalpingitis, epiglottis, pericardis, cellulitis, osteomyelitis,endocarditis, cholecystitis, intraabdominal infections, urinary tractinfection, mastoiditis, aortic graft infection, conjunctitivitis,Brazilian purpuric fever, occult bacteremia, chronic obstructivepulmonary disease and exacerbation of underlying lung diseases such aschronic bronchitis, bronchietasis and cystic fibrosis.

Compositions comprising inhibitors of H. influenzae type IV pilifunction are provided. The compositions may consist of one of theforegoing active ingredients alone, may comprise combinations of theforegoing active ingredients or may comprise additional activeingredients used to treat bacterial infections. As discussed above, thecompositions may comprise one or more additional ingredients such aspharmaceutically effective carriers. Also as discussed above, dosage andfrequency of the administration of the compositions are determined bystandard techniques and depend, for example, on the weight and age ofthe individual, the route of administration, and the severity ofsymptoms. Administration of the pharmaceutical compositions may be byroutes standard in the art, for example, parenteral, intravenous, oral,buccal, nasal, pulmonary, rectal, intranasal, or vaginal.

Animal Model

Methods of the invention may be demonstrated in a chinchilla modelwidely accepted as an experimental model for OM. In particular, achinchilla model of NTHi-induced OM has been well characterized(Bakaletz et al., Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes,Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki and Bakaletz,Infect. Immun., 62: 1710-1718, 1994; Mason et al., Infect. Immun.,71:3454-3462, 2003), and has been used to determine the protectiveefficacy of several NTHi outer membrane proteins, combinations of outermembrane proteins, chimeric synthetic peptide vaccine components, andadjuvant formulations against OM (Bakaletz et al., Vaccine, 15: 955-961,1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999; Kennedy etal., Infect. Immun., 68: 2756-2765, 2000; Kyd et al., Infect. Immun.,66:2272-2278, 2003; Novotny and Bakaletz, J. Immunol., 171, 1978-1983,2003).

In the model, adenovirus predisposes chinchillas to H.influenzae-induced OM media, which allowed for the establishment ofrelevant cell, tissue and organ culture systems for the biologicalassessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993;Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infectionalone has been used to assess the transudation of induced serumantibodies into the tympanum (Bakaletz et al., Clin. Diagnostic LabImmunol., 4(2): 223-5, 1997) and has been used as a co-pathogen withNTHi, to determine the protective efficacy of several active and passiveimmunization regimens targeting various NTHi outer membrane proteins,combinations of OMPs, chimeric synthetic peptide vaccine components, andadjuvant formulations as vaccinogens against otitis media (Bakaletz etal., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect.Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4):2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).

BRIEF DESCRIPTION OF DRAWING

FIG. 1 provides the timeline of the immunization regimen, viralinoculation, bacterial challenge, and OM disease assessment period forthe efficacy experiments described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention wherein Example 1describes recombinant production of chimeric proteins of the invention,Example 2 describes assays to test the immunogenicity of the chimericproteins of the invention, Example 3 describes assays for evaluatingpassive immunization, Example 4 describes assays for evaluating activeimmunization and Example 5 describes the evaluation of a chimericprotein of the invention.

Example 1 Synthesis of Chimeric Proteins

The chimeric proteins of the invention were produced using standardrecombinant methods. Initially, a gene-synthesis company, (Blue HeronBiotechnology Inc.) was contracted to make the initial plasmid based onthe chimeric protein amino acid sequences described herein that wereoptimized for E. coli preferred codon usage. Briefly, the native NTHipilin protein sequence was modified by truncating the N-terminus(residues 1-39 of SEQ ID NO: 2) and adding a HIS-tag sequence and athrombin cleavage site as set out in SEQ ID NO: 3. The HIS-tag waspreceded by a sequence (MUSS) to assist in expression. The thrombincleavage site allowed for release of the HIS-tag. These plasmids werethen cloned into the E. coli expression vector pET-15b vector (Novagen).The plasmid were then transformed into E. coli strain “Origami(DE3)”(available from Novagen) as the host for expression of solubleHis-tagged chimeric proteins. Another E. coli host cell expression stainthat may be used is Origami B(DE3) (Novagen).

The His-tagged variants of the chimeric proteins will be recovered bynickel column chromatography, then used for initial studies to determineif they are reactive with antisera directed against any of thefollowing: native OMP P5-fimbrin, LB1 (full length 40 amino acidpeptide), LB1(1) (a synthetic peptide representing just the 19 aminoacid B-cell epitope of LB1), recombinant PilA protein or native PilAprotein. Once the His-tag is removed by thrombin site cleavage, therecombinant chimeric proteins will be used as immunogens to determinetheir immunogenicity and protective capability.

Exemplary chimeric proteins of the invention have the sequences as setout in Table 2 below. The chimeric proteins having the amino acidsequences of SEQ ID NOS: 10, 12 and 14 have been expressed by E. coli asdescribed above.

TABLE 2 SEQ ID NO: Chimeric Protein Amino Acid Sequence  9MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCRSDYKFYEDANGTRDHKKGCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDA SLFPANFCGSVTQ 10MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCLVRSDYKFYEDANGTRDHKKGHTCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCK GTDASLFPANFCGSVTQ 11MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCRSDYKFYEDANGTRD HKKGCGSVTQ 12MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCLVRSDYKFYEDANGTR DHKKGRHTCGSVTQ 13MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQRSDYKFYEDANGTRDHKKG 14MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQLVRSDYKFYEDANGTRDHKKGRHT 15MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCRSDYKLYNKNSSSNSTLKNLGECTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGT DASLFPANFCGSVTQ 16MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCLVRSDYKLYNKNSSSNSTLKNLGERHTCTGGKNGIAADYITAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWT TCKGTDASLFPANFCGSVTQ 17MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCRSDYKLYNKNSSSNST LKNLGECGSVTQ 18MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCLVRSDYKLYNKNSSSN STLKNLGERHTCGSVTQ 19MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQRSDYKLYNKNSSSNSTLKNLGE 20MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQLVRSDYKLYNKNSSSNSTLKNLGERHT21 MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCRSDYKLYNKNSSSLKNLGECTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDAS LFPANFCGSVTQ 22MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCLVRSDYKLYNKNSSSTLKNLGERHTCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTC KGTDASLFPANFCGSVTQ 23MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCRSDYKLYNKNSSTLKN LGECGSVTQ 24MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNEILNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYTTQATGNAATGVTWTTCLVRSDYKLYNKNSSTL KNLGERHTCGSVTQ 25MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQRSDYKLYNKNSSTLKNLGE 26MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQLVRSDYKLYNKNSSTLKNLGERHT 27MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCRSDYKFYDNKRIDCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANF CGSVTQ 28MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCLVRSDYKFYDNKRIDRHTCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASL FPANFCGSVTQ 29MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCRSDYKFYDNKRIDCGS VTQ 30MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCLVRSDYKFYDNKRIDR HTCGSVTQ 31MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQRSDYKFYDNKRID 32MGSSHHHHHSSGLVPRGSHMTKKAAVSELLQASAPYKADVELCVTSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTCKGTDASLFPANFCGSV TQLVRSDYKFYDNKRIDRHT

Additional exemplary chimeric proteins of the invention have the aminoacid sequences as set out in Table 3 below. These chimeric proteins havebeen expressed by E. coli and purified using a HIS-tag, as describedabove. The chimeric proteins set out in Table 3 have the His tagsequence removed for use as an immunogen. The chimeric protein havingthe amino acid sequence of SEQ ID NO: 56 was used in the studiesdescribed in Example 5.

TABLE 3 SEQ ID NO: Chimeric Protein Amino Acid Sequence 54GSHMTKKAAVSELLQASAPYKADVELCLVRSDYKFYEDANGTRDHKKGRHTCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGS VTQ 55GSHMTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCLVRSDYKFYEDANGTRDHKKGRHTCGSVTQ 56GSHMTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQLVRSDYKFYEDAN GTRDHKKGRHT 57GSHMLVRSDYKFYEDANGTRDHKKGRHTGPSLKLTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCK GTDASLFPANFCGSVTQ

Example 2 Immunogenicity of Chimeric Proteins

Rabbits or chinchillas are immunized with the chimeric proteins. Rabbitsreceive an initial immunizing dose of 500 μg of a chimeric protein incomplete Freund's adjuvant. The rabbits receive a second dose of 400 μgof the chimeric protein 21 days later. The rabbits receive a third doseof chimeric protein in complete Freund's adjuvant 42 days after theinitial immunizing dose with 400 μg of the same peptide in either IFA orPBS (one rabbit per diluent). Sera are obtained 3 weeks after each dose.Chinchillas receive an initial immunizing dose of 10 μg of the chimericprotein in the adjuvant monophosphoryl lipid A (MPL). One month (˜30days) later, chinchillas receive a second identical dose. The third andfinal dose is delivered 30 days after the second dose. Sera are obtained˜10-14 days after each dose. The sera from all animals are assessed fortiter and specificity against the LB1 peptide (40-mer), LB1(1), PilAprotein and the chimeric proteins, by ELISA, Western blot and biosensor.Antisera are also tested against whole bacteria via flow cytometry(FACS) analysis.

Example 3 Evaluating Passive Immunization

The protection conferred by an animal's immune response directed againstthe chimeric proteins of the invention is determined in a chinchillamodel of experimental otitis media. Chinchillas are passively immunizedwith 5 ml/kg hyperimmune chinchilla or human serum directed against achimeric protein of the invention. Control chinchillas receive normalchinchilla serum or normal human serum. Next the chinchillas receivefirst a viral co-pathogen intranasally, then a week later, an intranasalchallenge with the NTHi bacteria. The chinchillas are examined and rateddaily or every 2 days for up to 35 days after bacterial challenge.Immunized chinchillas receiving immune chinchilla or human serum displayreduced tympanic membrane pathology and reduced or absent signs ofinfection of the middle ear space as determined by both video otoscopyand tympanometry. In this assay, the presence of middle ear fluids inchinchillas receiving chinchilla or human anti-chimeric protein serum isreduced when compared to controls.

Example 4 Evaluating Active Immunization

Cohorts of 5-10 chinchillas each are actively immunized with either asaline control preparation, an adjuvant-only preparation, or one of thechimeric proteins of the invention that has been admixed with anappropriate adjuvant. The immunogens are assessed for endotoxin contentprior to their use as an immunogen via a chromogenic Amoebocyte Lysateassay which is commercially available from Whittaker Bioproducts underthe designation QCL-1000. The chinchillas are then subcutaneouslyinjected with 10 μg immunogen in the adjuvant MPL (or anotherappropriate adjuvant). Then 30 days later they receive 10 μg of the sameimmunogen in MPL. Thirty days following the second immunization, theseanimals receive the final immunizing dose. Approximately 10-14 daysafter the final immunizing dose is delivered, chinchillas are challengedboth transbullarly and intranasally with a strain of NTHi. Thechinchillas are assessed over a 35-day period for: tympanic membranepathology by video otoscopic examination and tympanometry;semiquantitation of NTHi recovered via epitympanic tap of the inferiorbulla and passive lavage of the nasopharynx; and light microscopicexamination of fixed middle ear mucosal epithelium and tympanic membranefor histopathology. For example, chinchillas immunized with the chimericproteins of the invention will have reduced tympanic membrane pathology,will be free of middle ears effusions or they will contain effusionsthat are culture-negative, there will be reduced or no biofilm presentin the tympanum and there will be minimal thickening of the middle earmucosa, minimal osteoneogensis and reduced presence of both red bloodcells and inflammatory cells in the subepithelial space.

Example 5 Evaluation of Chimeric Proteins

The protective efficacy of the chimeric protein having the amino acidsequence of SEQ ID NO: 56 (referred to as “chim-V3” herein) wasevaluated using the chinchilla passive-transfer, superinfection model ofOM. This chimeric peptide comprised the B-cell epitope of the LB1peptide (SEQ ID NO: 5) expressed after the C-terminal glutamine residueof recombinant PilA (residues 40-149 of SEQ ID NO: 2). To generatepolyclonal antiserum for use in passive transfer efficacy studies, thechim-V3 protein was delivered to a cohort of adult chinchillas with theadjuvant, monophosphoryl lipid A (MPL) plus trehalose dimycolate(Corixa). A timeline depicting the immunization regimen is set out inFIG. 1. To generate immune serum pools, alert prone chinchillas wereimmunized subcutaneously 3 times with 30 μg of chim-V3 plus 10 μg of MPLor 10 μg MPL alone every 21 days. At day 56, a terminal bleed of theinoculated animals was collected and serum was pooled for transfer tonaïve juvenile animals. To study efficacy, a separate cohort of juvenilechinchillas was first challenged with adenovirus on day −7. Six dayslater (day −1), the pooled anti-chim-V3 immune serum was passivelytransferred to these adenovirus-compromised animals. The following day(day 0), animals that received anti-chim-V3 serum by passive transferwere challenged with the bacterium, nontypeable Haemmophilus influenzae.These animals were then monitored for incidence and severity of diseaseover a 26-day time-course (relative to bacterial challenge) by dailyvideo otoscopy and tympanometry as well as Xenogen in vivo imaging everyother day.

The titer of anti-chim-V3 antibody was measured in the immune serumcollected from the inoculated animals using an ELISA. This analysisdemonstrated that the collected antiserum contained antibodies specificfor the chim-V3 protein. The presence of anti-chim-V3 antibodies in thecollected antiserum as also confirmed using Western blot analysis.

FACS analysis was used to measure the ability of serum immunoglobulinsfrom immunized animals to recognize surface exposed native structuresexpressed by NTHi 86-028 NP. NTHi bacteria were incubated with chim-V3antiserum, washed, then incubated with naïve or immune FITC-Protein A,washed and analyzed by FACS analysis. Inoculation with the chim-V3protein induced a significant increase in antibodies that were capableof recognizing the NTHi surface proteins or chim-V3 protein. The dataobtained were dependent on both antibody titer and avidity as well asrelative expression of both the type IV pilus and the OMP P5-homologousadhesion by NTHi when grown in vitro.

The luminescent reporter NTHi 86-028 NP pKMLN-1 was used to detect NTHiinfection in the animals inoculated with chim-V3 protein using Xenogenin vivo real-time imagining. Growth curves of the luminescent strainNTHi 86-028 NP pKMLN-1 and the parental strain NTHi 86-028 demonstratedthat growth of the luminescent NTHi strain was comparable to theparental strain. Luminescent imaging of NTHi residing in the nasopharynxof the inoculated animals was readily accomplished however, due to themicroaerophilic nature of the diseased middle ear, luminescence of NTHipresent in the middle ear could not be monitored over the entire diseasecourse because the luminescence is dependent on the availability ofoxygen. Animals were monitored every other day for the presence ofluminescent bacteria, and if bacteria were detected, this was recordedas a luminescent event. Luminescent infection was detected at least sixdays after challenge in the inoculated animals. The total number ofluminescent events in the chim-V3 inoculated animals was less than thetotal number of luminescent events in the control animals (inoculatedwith MPL only).

Throughout the course of the study, daily video otoscopy andtympanometry was used to determine the percent of chinchilla middle earswith OM. Inoculation with chim-V3 caused 53% reduction in the number ofanimals with middle ears having OM as compared to control animals(inoculated with MPL only).

All of these studies demonstrate that the chim-V3 protein wasimmunogenic and anti-chim-V3 antibodies were protective in thechinchilla passive transfer-superinfection model of OM.

1. A chimeric protein comprising a portion of PilA and a portion of theLB1 peptide
 2. The chimeric protein of claim 1 wherein the portion ofthe LB1 peptide comprises the B-cell epitope of the LB1 peptide.
 3. Thechimeric protein of claim 2 wherein the portion of the LB1 peptidecomprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO:
 8. 4. The chimeric protein of claim 1 comprising theamino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 32, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56.5. A polynucleotide encoding the chimeric protein of claim
 1. 6. Avector comprising a polynucleotide of claim
 5. 7. A compositioncomprising a chimeric protein of claim 1 and a pharmaceuticallyacceptable carrier.
 8. An antibody that specifically binds to a chimericprotein of claim
 1. 9. A composition comprising an antibody of claim 8and a pharmaceutically acceptable carrier.
 10. A method for eliciting animmune response to NTHi bacteria comprising administering an immunogenicdose of one or more chimeric proteins of claim 1 to a patient at risk ofNTHi bacterial infection.
 11. The method of claim 10 wherein the NTHiinfection is in the middle ear, nasopharynx or lower airway.
 12. Amethod of treating or preventing NTHi bacterial infection comprisingadministering an antibody of claim 8 to a patient in need thereof. 13.The method of claim 12 wherein the NTHi infection is in the middle ear.14. The method of claim 10 wherein the NTHi bacterial infection involvesa pathological condition selected from the group consisting of otitismedia, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis,septic arthritis, meningitis, sepsis, salpingitis, epiglottis,pericardis, cellulitis, osteomyelitis, endocarditis, cholecystitis,intraabdominal infections, urinary tract infection, mastoiditis, aorticgraft infection, conjunctitivitis, Brazilian purpuric fever, occultbacteremia, chronic obstructive pulmonary disease, chronic bronchitis,bronchietasis and cystic fibrosis.
 15. The chimeric protein of claim 1,wherein the portion of NTHi PilA protein comprises residues 40-149 ofSEQ ID NO:
 2. 16. The chimeric protein of claim 15 wherein the B-cellepitope of LB1 is inserted before residue 40 of SEQ ID NO:
 2. 17. Thecomposition of claim 7 wherein the composition is formulated for a routeof administration selected from the group consisting of parenteral,intravenous, oral, buccal, nasal, pulmonary, rectal, intranasal,transdermal, subcutaneous, intramuscular or vaginal administration.